Solid state fermentation

Details Solid state fermentation Solid state fermentation

2000-05-05

2003-12-16

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C435S244000, C435S286700, C435S291300, C435S291600, C435S294100, C435S261000

Reexamination Certificate

active

06664095

ABSTRACT:

BACKGROUND
Solid state fermentation has been practiced for centuries, most often in connection with food production, and can be defined as a technique for growing microorganisms, such as fungi, yeast and bacteria, on moist solid substrates. In recent years, there has been a resurgence of interest in solid state fermentation and its applicability to the production of enzymes, metabolites and organic compounds. Solid state fermentation devices provide several advantages over the commonly used process of submerged fermentation in product yield, cost and ease of use. Despite their economic advantages, the commercialization of solid state fermentation devices for industrial use has been limited for lack of efficient and practical designs.
A wide variety of solid state fermentation devices have previously been described (for review see, Larroche et al., “Special Transformation Processes Using Fungal Spores and Immobilized Cells”, Adv. Biochem. Eng. Biotech., (1997), Vol 55, pp. 179; Roussos et al., “Zymotis: A large Scale Solid State Fermenter”, Applied Biochemistry and Biotechnology, (1993), Vol. 42, pp. 37-52; Smits et al., “Solid-State Fermentation-A Mini Review, 1998), Agro-Food-Industry Hi-Tech, March/April, pp. 29-36). These devices fall within two categories, those categories being static systems and agitated systems. In static systems, the solid media is stationary throughout the fermentation process. Examples of static systems used for solid state fermentation include flasks, petri dishes, trays, fixed bed columns, and ovens. Agitated systems provide a means for mixing the solid media during the fermentation process. One example of an agitated system is a rotating drum (Larroche et al., supra).
A major problem in both static and agitated solid state fermentation systems is obtaining efficient removal of heat that is generated during the fermentation process. One method of heat removal employed by numerous solid state fermentation systems is aeration. The disadvantage of using aeration as the means of heat removal is that not only is heat removed, but water is also evaporated from the solid matrix, leading to desiccation of the substrate. Constant aeration also makes it more difficult to maintain a stable environment inside the bioreactor with respect to oxygen and carbon dioxide concentrations. Another means of avoiding heat build up is mixing the substrate bed. Unfortunately, mixing during fermentation leads to damage of the cells and gross aggregation of substrate particles. Aggregation of substrate leads to inhomogeneities in local substrate temperature resulting in local differences in biomass growth and activity. These problems are compounded in the large scale systems often required for industrial preparation of certain products. The large scale practice of solid state fermentation using the devices available in the art has the additional disadvantage of being labor intensive.
The steps involved in solid state fermentation include, 1) sterilization of the cultivation device and the cultivation media, 2) inoculation of the cultivation media with the microorganisms, 3) cultivation of the microorganisms 4) extraction of biological products from the cultivated microorganisms, and 5) post extraction treatment of the waste materials and the cultivation device. It is also desirable that the cultivation system provide a mechanism whereby the growth environment during the cultivation process is precisely controlled such that specified conditions are maintained throughout the cultivation process. None of the devices available for solid state fermentation to date provide for carrying out all of the steps required for solid state fermentation in a single fermentation device. Up to now, the practice of solid state fermentation has involved carrying out multiple manipulations which are both tedious and impractical. Such manipulations often risk exposing the cultivation environment to contaminants from outside the cultivation environment, preclude the ability to efficiently and precisely control the cultivation, and lead to reduced product quality and/or yield.
There exists a need for a compact reactor that combines all the operations involved in solid state fermentation into a single device capable of operating in a contained manner and controlling the environment within the bioreactor without inhibiting growth of the microorganism. Furthermore, there exists the need for a device that will allow homogeneous addition of chemicals and nutrients to a bioreactor without contamination.
SUMMARY OF THE INVENTION
The present invention provides an improved solid state fermentation device for the cultivation of microorganisms. In general, the invention provides a bioreactor and a process for using the bioreactor for the cultivation of microorganisms on solid media. The Applicants herein refer to the bioreactor of the present invention as a PLAFRACTOR™. In preferred embodiments, the invention provides a bioreactor that is modular in nature and carries out all of the processes of solid state fermentation in a single, contained environment. The modular nature of the bioreactor allows the size of the bioreactor to be adjusted to suit the user's need. The construction of the bioreactor allows solid state fermentation to be carried out in a manner such that the fermenting microorganisms are kept isolated from the outside environment during the course of the fermentation process. In certain preferred embodiments, the environment inside the modules is precisely controlled to meet specified conditions.
One aspect of the bioreactor is a mechanism to remove heat that accumulates inside the bioreactor during fermentation by conduction. Specifically, the bioreactor is constructed by stacking individual modules. The modular construction of the bioreactor provides multiple modules stacked on top of one another, each with a base connected to a frame for holding the solid medium in isolation from the exterior environment. The base plate of the bioreactor has multiple channels, called non-communicating channels, that carry heating and cooling fluids sandwiched between two sheets. Heat is transferred to and from the modules by conduction. In this way the temperature of the module is precisely maintained to meet the specific requirements of different microorganisms.
Another aspect of the bioreactor is a mechanism to add fluids to the interior of the modules. In this embodiment, the base of the module mentioned above contains a second set of channels, called communicating channels, to deliver fluids to the inside of the module thereby providing a way to adjust the moisture and oxygen content within the bioreactor. For example, for optimum growth, some microorganisms require high carbon dioxide concentrations. Another aspect of this embodiment provides a mechanism by which compounds of interest can be extracted from the microorganisms. For example, extracting fluids can be sent through the communicating channels for extracting the compound of interest for collection. In yet another aspect of this embodiment, the communicating channels can send steam, gas (e.g., ethylene oxide or ozone), or chemicals (e.g., beta-propiolactone, hydrogen peroxide or pyrocarbonic acid diethyl ester) into the bioreactor for sterilization of the bioreactor and its contents before and after fermentation. A final aspect of the present invention is that materials, (e.g., chemicals and/or nutrients), can be added to the bioreactor while the bioreactor is in operation.
In a further embodiment, the present invention provides a mechanism to mix the contents of the bioreactor. According to the present invention the interior of each module has a mixing arm that revolves about the central axis of the module while rotating. Mixing can be carried out at any point in the fermentation process that mixing is deemed to be appropriate. Preferably, mixing occurs after inoculation of the media inside the bioreactor to evenly distribute the inoculum into the media within the bioreactor.
The teachings of the present invention are particularly applica

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